The present invention relates to a novel pharmacological use of Anapsos. Anapsos is a neutral extract of Polypodium comprising 118 milligrams of a water soluble extracts and 2 milligrams of a lipid soluble fraction.
The present invention describes the involvement of the Anapsos in the regulation of the expression of the adhesion molecules. Anapsos reduces the expression of the alpha chain of integrins xcex2-2 (CD11a and CD11b), the beta chain of integrins xcex2-2 (CD18), and the differentiation antigen CD54 of the immunoglobulins superfamily. It is also capable of normalizing alterations of the immunological phenotype.
Further, is described the use of Anapsos in any diseases where an excess of inflammation, derived from cellular and tissue lesions, is responsible for pathologically symptoms and indications.
The cells of the immune system are in contact with other cells and the extra-cellular matrix in order to efficiently perform their required functions. This is due to that they must be able to recognize the situation of their surroundings. Accordingly, the leukocytes not only have specific surface receptors capable of being specifically activated in response to determined stimuli, but also comprises a number of molecules which globally are called adhesion molecules. The adhesion molecules act as receptors for ligands which are situated on other cells and as receptors capable of binding amino acid sequences present in different extracellular matrix proteins, such as collagen, fibronectin, lamina, and others. The adhesion molecules, beside being involved in the cellxe2x80x94cell and cellxe2x80x94extracellular matrix adhesion, collaborate in the cellular activation by sending co-activator signals into the interior of the cell.
The migration of leukocytes to the tissue, essential for the immunological response, is mediated by a number of molecular interactions where the adhesion molecules play a fundamental role. The adhesion molecules are classified into 3 structural based categories:
the selectins
the family of integrins
the superfamily of immunoglobulins
In the first step of inflammation, the leukocytes acumulate around the endothelial wall causing the endothelial cells to remove themselves from each other. This initial process is mediated by the interaction of specific endothelial selecting (selectin E and P) and their corresponding leukocyte receptor (sLex) and between leukocyte selectin L and specific adhesion molecules of the endothelium. Simultaneously, with the expression of the adhesion molecules, are also released pro-inflammatory cytokines. Following, an activation signal induces a conformational change in the extra-cellular domains of the leukocyte integrins which gives a stronger adhesion. This is mediated by interactions between specific integrins and their ligands (LFA-1/ICAM-1, VLA-4/VCAM-1). As a consequence of the leukocyte/endothilium adhesion the accumulation of leukocytes is reduced and there are produced extravasation and migration of the leukocytes, from the blood circulation to the focus of inflammation, by chemotaxis. Consequently, the adhesion molecules are responsible for different processes of adhesion, mediating the final adhesion to the endothelium, the extravasation, and the migration towards the focus of inflammation.
The alpha and beta Chains of the integrins xcex2-2 (CD11a, CD11b, CD18) are extended throughout all tissues. Consequently, a decrease in their expression gives an anti-inflammatory effect in the tissues. The adhesion molecule CD54 belongs to the immunoglobulin superfamily and is also highly distributed in various tissues, such as endothelium, leukocytes, etc.
In patients with multiple sclerosis there has been observed an increase in the lymphocyte population CD4+CD29+CD45RA. Further, recent data from the literature of virgin and memory cells indicates that revertant CD4+CD29+CD45RA+ cells are of fundamental importance as these are the authentic memory cells. They have a longer half-life as compared to CD45RO+ and once activated capable of being maintained years in the organism.
Inflammation, which might well be a normal physiological process, is when it is taken to its extreme converted into a pathological issue. For example, this happens in the major part of the auto-immune processes (systematic or organ specific), chronic inflammatory diseases, or infections which symptoms are characterized by an exaggerated inflammation giving rise to the corresponding damage of organs or tissues. Consequently, any medicament capable of decreasing the expression of the adhesion molecules, which under inflammation processes normally have an increased expression, may be suitable for the treatment of these diseases, independently if the aetiological cause of the specific disease. Such disease might be neuro-degenerative disorders (multiple sclerosis, Alzheimer), and connective tissue diseases (systematic lupus eritematose, Sjxc3x6gren syndrome, reumatoide arthritis, Behcet disease, etc).
The anti-inflammatory action, due to reduction of the expression of the adhesion molecules, is performed independently of the inhibition-stimulation of the inflammatory cytokines and the stimulatory effect of the cellular immunity (increase of TH1-like cytokines and increase of T CD8+ lymphocytes and NK cells).
The extracts of the genus of the Polypodiaceae family have traditionally been used in Central America in the popular medicine attributing to it different activities such as: anti-inflammatory activity, Boletin de la Sociedad Quimica del Perxc3xa pag 91 (1988); prevention of tumor malignant, Nature 214: 1256-1258 (1967). There have been described clinical effects in diseases related to immunological deficits, such as atopic dermatitis; Dermatolxc3x3gica 173: 154-156 (1986); atopic dermatitis, Allergology et Inmunopathology 15: 185-189 (1987); International Journal of Dermatology 13: 276-282 (1974); Planta Mxc3xa9dica 58: 306-310 (1992) and vitiligo, International journal of Dermatology 28: 479 (1989). In these publications it has been identified that the extracts of Polypodium leucotomos have activity in relation to hyperqueratosis, paraquerotosis, epidermal mitosis, and lesions of the epidermis.
The extracts of these ferns have been described to have immuno-modulatory capacity in patients with atopic dermatitis, giving a normalization of the CD4+/CD8+ relation after treatment with extract of Polypodium leucotomos (Anapsos(copyright)), Dermatxc3x3logica 173:154-56 (1986); Annals Inmulogie 134:393-400 (1983). Annals of Psychiatry 3: 329-341 (1992) describes that the Anapsos(copyright) improve the memorizing, Decrease the levels of cytokines IL-1xcex2-2 and IL-2 in the frontparietal cortex and decrease IL-1xcex2 in hypo-campus, Br. J. Clin. Pharmacol 43: 85-89 (1997) describes the immuno-modulatory effect in vitro of the polar extract of Polypodium leucotomos (Anapsos(copyright)) in relation to the cytokines IL-1xcex2, IL-2, IL-10, INF-(which could give a pleiotropic effect in the different populations of the immune system.
Concerning the patent literature following documents of relevance have been identified.
The European patent application EP-503.208 describes a process for obtaining a water-soluble extract by extraction of the leaves and/or rhizomes of different ferns. This document specifies that these extracts have immunological activity and consequently useable in diseases involving a depression of the immune system, generally with a deficit of T suppressor lymphocytes, and with beneficial affects in the auto-immuno diseases and viral infections. Examples of pathological utilities are: reumatoide arthritis, lupus eritematose, syndrome of Sjxc3x6gren, multiple sclerosis, hepatitis B, syndrome of Di George, auto-haemolytic anaemia, atopic dermatitis, psoriasis, Basedow disease, Chron disease, mistenia, vitiigo, herpes zoster, etc. The extract increases the level of T-suppressor lymphocytes.
The Spanish patent ES-2.088.770 teaches a pharmaceutical composition based on a water-soluble and a lipid soluble fraction of the leaves and/or rhizomes of different ferns with beneficial effects against cognitive dysfunctions, and/or neuroimmunes, in particular for the treatment of Alzheimer disease.
The U.S. Pat. No. 5,614,197 describes the use of extracts of Polypodium as photo-protector agents and as anti-oxidants. The Spanish patent ES-470.204 relates to natural terpenes with anti-psoriasis activity which are obtained by extraction of the rhizomes and leaves of different ferns. The Spanish patent ES-490.293 relates to a medicament with anti-inflammatory effects in pathological problems which affects the osteolocomotor apparatus of the human organism, specially the arthritis. The medicament is obtained as extracts from fern of the family Polypodiaceae using both the leaves and the rhizomes. The U.S. Pat. No. 3,839,553 uses the extracts of Polypodium as capillary conditioners. The French patent FR-2.395.266 relates to obtaining a a-d-gluco-octane-delta-l-lactone-eno-diol and its calcium salt starting from the ferns of Polypodium, having immuno-suppressing and antiviral activity in mammalians.
The patent application having the publication number WO-97/40838 describes the use of a sulfur lipid for the treatment of inflammatory disorders of the skin, specially in psoriasis, by inhibition of the plaque aggregation factor. The sulfur lipid is obtained from leaves by methanol extraction.
The Polypodium extracts as described in the art are water-soluble or hydrophilic extracts which may be obtained by extraction with polar solvents followed by different purification steps such as purification by resins via inter-chancing of ions, absorption over active carbon followed by evaporation of the solvents or lyophilization. Corresponding, the lipid or lipid soluble fractions may be obtained by extraction with apolar solvents such as hexane, chloroform, or ether to obtain the different triterpenes present in the leaves and/or rhizomes. The pharmaceutical composition of the present invention comprises as active ingredient the extract of Polypodium, Anapsos, corresponding to the pharmaceutical composition as described in the Spanish patent ES-2.088.770. It comprises a water-soluble and a lipid soluble fraction and pharmaceutically acceptable carries. Each unit dose consist of 120 mg extracts, wherein 118 mg is a water-soluble fraction, equivalent to 60 mg of alcohol extract, and 2 mg is a lipid soluble fraction.
Suitable excipients are the ones as normally used in the pharmaceutical industry such as a lactose preparation, starch, magnesium stearate, silicium dioxide. It may be possible to use other excipients and in other proportions.
The water-soluble extract is obtained by maceration, in water for 24-48 hours, the leaves and rhizomes of the ferns of Polypodium aureum, Polypodium leucotomos, Polypodium vulgare, Polypodium trisereiale, Polypodium aquilinum, Drypteris crassirhizoma, or Cyathe taiwamiana. The extract is characterized by the presence of quinine acid, malic acid, lactic acid, citric acid, fumaric acid and by the absence of any kind of sulfur lipid. The lipid soluble fraction is characterized by the presence of Neo-hop-13(18)-eno, Fern-9-(11)-eno, and Hop-(22)-29-eno as identified via mass spectrometry.
The documents of the prior art describe different activities, sometimes in an empirical manner without specifying the mechanism, of the polar and apolar extracts of the rhizomes and/or leaves of the ferns of the Polypodiaceae family.
The pharmacologically actions may be summarized as:
Immuno-modulatory activity in diseases with a deficit in T-suppressor lymphocytes, infectious and autoimmunes, where the extracts exhibit an pleiotropic effect over the different populations of cytokines,
Collagenpoyetic activity and application in psoriasis, atopic dermatitis,
Anti-inflammatory activity of the osteolocomotor apparatus, principally the arthritis,
Anti-inflammatory activity characterized by the inhibition of the plaque aggregation factor.
In none of the identified prior art documents it has been described that the extracts of the different ferns are capable of regulating the expression of the adhesion molecules nor that these are capable of regulating the lymphocyte CD4+CD29+CD45RA+ populations present in increased amounts in patients with multiple sclerosis.
The problem to be solved by the present invention is to provide a novel therapeutic application of the Anapsos based on the regulation of the different cellular mediators. The regulation of the expression of the adhesion molecules (the decrease of the expression of the chains of integrins and the immunoglobulin superfamily) and the normalization of the lymphocyte CD4+CD29+CD45RA+ population makes the Anapsos a medicament suitable for treatment of diseases which relate to an inflammatory process, in particular in the treatment of multiple sclerosis.
The present inventors have demonstrated, that the Anapsos (118 mg of a water-soluble extract and 2 mg of a lipid soluble fraction of Polypodium) has regulatory effects in relation to the expression of adhesion molecules, in mononuclear cells of the peripheral blood of healthy humans, both in vivo and in vitro. Further, it has been demonstrated that Anapsos has a capacity similar to azathioprine to recover the normality of the lymphocyte CD4+CD29+CD45RA+ population, present in increased amounts in patients with multiple sclerosis, thereby obtaining a stabilization of the patients.
At a dose from 0 to 5000 xcexcg/ml of Anapsos and using different doses of phytohaemagglutinin, the Anapsos in vitro is capable of inhibiting the increase of the expression of the adhesion molecules (CD54 and CD11b), as induced by the phytohaemagglutinin, in studies realized on mononuclear cells of human peripheral blood. The results are most significant at a dose of 150 xcexcg/ml of Anapsos and 10 xcexcg/ml of phytohaemagglutinin.
After administration of 720 mg of extract per day for 11 days in a human, the Anapsos decreases the percentage of the lymphocyte populations CD11a, CD11b, and CD54. Accordingly, the extract inhibits the expression of certain adhesion molecules of the integrins xcex22 (CD11a, CD11b) and of the superfamily of immunoglobulins (CD54).
Accordingly, beside the stimulating action on the cellular immunity as performed by the cytokines and its immuno-modulatory action as described in the art, the Anapsos has a strong anti-inflammatory capacity, similar to phenylbutazone, used as a control in anti-inflammatory studies in rats. The anti-inflammatory effects of Anapsos has not been directly related to its capacity of regulating the expression of the adhesion molecules.
A pharmaceutical composition based on water-soluble and lipid soluble extracts of Polypodium has, in clinical studies performed on humans, been demonstrated to be effective against some diseases which involve inflammatory processes such as multiple sclerosis, prostatitis, and pharyngitis. Between them, these diseases have a different aetiological cause.